Composition and method for manufacturing ion selective electrode sensors

ABSTRACT

This invention pertains to fluorophoric compositions of a 7-amino-coumarin derivative and methods of their use for enhancing visualization of various constituents of ion selective electrodes.

This is a division of application Ser. No. 08/668,223, filed on Jun. 21,1996, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains to optical inspection of productsproduced during automated manufacturing. More particularly, thisinvention relates to a method for tagging semi-transparent polymericlayers contained in a multilayer sensor structure in order to facilitateoptical inspection and alignment of the layers.

2. Description of the Related Art

In the health care field, particularly in the area of clinicaldiagnostics, ion-selective-electrode (ISE) sensors are commonly used tomeasure the activity or concentration of various ions and metabolitespresent in biological fluids. ISE sensors employ potentiometric oramperiometric electrochemical processes which generate potential orcurrent signals that are related to the activity of an ion of interestin a sample. For example, ISE sensors are typically used to determinechloride, potassium, lithium, calcium, magnesium, carbonate, hydrogen,and sodium ion content in such fluids. Generally, the signal generatedwithin the sensor is linearly dependent on the logarithm of the activityof the ion of interest for potentiometric analyses. The activity of anion of interest is defined as its concentration multiplied by anactivity coefficient, where the activity coefficient is generally knownor is available in the art.

Typically, solid state ISE sensors use a solid membrane as a sensingelement or electrode, the membrane being highly selective to the ionicspecies being sought and reacting to the ionic species with changes inionic conductivity. In addition, conventional ISE sensors may contain aninternal reference electrode. In operation, one surface of the sensingmembrane is immersed in a biological sample solution of ions for whichit is selective whereby a potential develops across the membrane surfaceat the interface of the solution and the membrane. In a potentiometricsensor, this potential varies with the concentration of ions in solutionand its magnitude is measured as a voltage. By comparing the voltagegenerated at the sensing membrane surface with that generated by areference electrode using a reference ionic solution, it is possible tocalculate the concentration of the ionic species being sought. Thedesired selectivity is often achieved by incorporating into the membraneof an ion-selective electrode an ion selective agent such as anionophore to increase the permeability of cell membranes to a specificion. Generally, ion-selective membranes are formed from a heavilyplasticized polymer matrix, such as polyvinyl chloride, which containsthe ionophore selective for the ion of interest. For example, theionophore valinomycin has been incorporated into a layer of membraneselective for potassium ions; trifluoroacetyl-p-butylbenzene or othertrifluoroacetophenone derivatives have been used as ionophores selectivefor carbonate ions.

By "biological sample" is meant any fluid of biological origin includingfluids of biological origin which have been chemically and/or physicallytreated, diluted, or concentrated prior to analysis. Examples ofbiological samples include serum, urine, plasma, whole blood,cerebrospinal fluid, amniotic fluid, saliva and tears.

A general discussion of the principles of ISE sensors is provided byForeman et al., "Ion Selective Electrodes", Automatic Chemical Analysis,Ellis Horwell Ltd., Chichester, England (1975). ISE sensors can beclassified according to the nature of the membrane material, and includesolid state membrane electrodes, glass membrane electrodes, liquidmembrane electrodes having charged ion-selective agents, and neutralliquid membrane electrodes having membranes formed from an organicsolution containing an electrically neutral, ion-selective agent such asan ionophore held in an inert polymer matrix.

Conventional ISE sensors are typically bulky and tend to require anundesirable large volume of biological fluid. For these reasons, muchattention has been directed towards developing ISE sensors of smallersize. These relatively smaller ISE sensors can be inexpensively massproduced using techniques similar to those employed in the manufactureof multilayer electronic components, which techniques include forexample, photolithography and screen printing as described in U.S. Pat.No. 4,454,007. Such ISE sensors may be in the form of a disposablecartridge or sensor assembly for use in an chemical analyzer and aregenerally produced on a planar substrate with plural reference elementsand plural sensor elements formed thereon. Electrical contacts arepositioned on the substrate face for each element, and a flow channel istypically positioned over the substrate reference and sensor elements todirect the sample being analyzed over the sensor elements. Liquidconduits are adapted to supply biological samples to the flow channeland to remove them from the ISE sensor device. The ISE sensor substrateis advantageously chosen to be of a structurally rigid material thatexhibits negligible distortion when pressure is applied from the flowchannel member, and is an electrical insulator to provide support forthe layers of a multilayer ion sensor. A preferred material for thesubstrate is alumina.

The manufacture of a complete ISE sensor includes numerous sequentialmanufacturing steps in which the patterns within adjacent layersproduced during successive manufacturing process must be alignedproperly with patterns produced by preceding processes. These successivemanufacturing steps are fairly complex in nature and may includeconventional screen printing using a screening or photographic mask, andfiring processes of paste-like compositions to produce the desiredperformance. The pattern and structure of the sensor resulting from eachstep of these many sequential operations must be accurately aligned withpatterns and structures generated in preceding process steps. Amisregistration of a single operation may result in an electrical openor short circuit in the finished sensor.

The use of finer and finer grids and closely spaced features as found inmodern ISE sensors requires a very high degree of positional accuracy ofimprinted features. Consequently, misregistration of pattern features onthe panels that may occur from misalignment of the panel and screeningmask or machine tool in any singular step of the multi-stepmanufacturing process is a particularly critical problem in contributingto manufacturing yields. Due to the importance of maintaining correctregistration of all the features generated by the sequence ofmanufacturing steps throughout the manufacturing process, modern ISEsensors must be inspected frequently at various stages of theirmanufacturing.

In order to improve the accuracy of inspection during sequentialmanufacturing of multilayer devices in mass produced quantities, machinevision systems are frequently employed. Machine vision systems acquirean image of a selected portion of the device through an electronicsensor and determine the existence of any extraneous features or marksin the image and the acceptability of any such marks by use of acomputer. The technology commonly employs a solid state CCD (chargecoupled device) or MOS (metal oxide semiconductor) type black and whiteor color television camera. Other components of a machine vision systemnormally include a lens that is attached to the television camera, aswell as mirrors, beam splitters (partially silvered mirrors that canreflect and transmit light at the same time), color filters, polarizers,etc. These additional components can be used to enhance contrast and/orto reduce the effect of unwanted information, to obtain the neededoptical geometric arrangement in a limited space, to acquire the image,to acquire and store a two-dimensional image, and to process and analyzethe image by some form of computer. Machine vision systems can alsoprovide important and accurate process control information to helpidentify "problem area(s)" of the sequential manufacturing process sothey may be corrected to improve quality and yield.

In the design of ISE sensors, many of the solid membranes that areselective to the ionic species being sought comprise a polymericmembrane composition that is essentially optically undistinguished froma surrounding or underlying material, i. e., is translucent, issemi-transparent, or has an optical reflectivity essentially similar tothat of the surrounding material. Consequently, the integrity, patternand outline of the imprinted membrane layer pattern may not be readilydifferentiated by machine vision systems. In such instances, it is knownto tag the material to be identified using fluorescent compounds thatare more readily capable of detection. The use of fluorescent techniquesto detect the presence of compounds is known in the art. Withfluorescent scanning, tagged samples are stimulated by a light beam atan excitation wavelength and the resulting stimulated fluorescentemission is examined. The stimulated fluorescent typically occurs at adifferent wavelength or wavelength band than the excitation wavelength.See, for example, U.S. Pat. No. 5,459,325, "High Speed FluorescentScanner", Hueton et als. A light source which is capable of emittinglight in the near-infrared illuminates the material to be inspected andan optical filter is used to select only the wavelengths emitted by thetagging fluorescing compound.

U.S. Pat. No. 4,983,817 relates to reading a luminescent andsubstantially transparent bar code on a background whose reflectance mayvary. Electrical signals corresponding to light reflections from bothluminescent and non-luminescent portions of the bar code are processedto provide a final signal that is decoded to provide the desiredreading.

U.S. Pat. No. 4,186,020 describes development of fluorescent inks thatcan be activated by ultraviolet light to fluoresce at longer wavelengthsin instances when the background fluorescence is less.

U.S. Pat. No. 5,095,204 presents a system and method for treating ormodifying bulk materials or formed articles such that they can be seenand identified under ultraviolet radiation without a permanentalteration of their appearance or properties.

U.S. Pat. No. 5,461,136 relates to a method for "marking" or "tagging" athermoplastic polymeric material by incorporating one or more nearinfrared fluorescing compounds therein and a method for separating orsorting a mixture of thermoplastic containers such as bottles. Alsoprovided are thermoplastic polymer compositions tagged with suchcompounds or residues as well as certain new compounds useful as nearinfrared fluorophoric markers.

However, a shortcoming in the application of conventional machine visionsystems to the inspection of sensor membranes is the degradation inperformance of the sensor membranes due to the inclusion of fluorescingcompounds to make them optically discernible. The selectivity andsensitivity of an ISE sensor membrane are critically dependent upon itschemical constituents and their relative balances. Consequently, therehas developed a need for a chemically inert ingredient such as afluorophore which may be added to the sensor membrane in a quantity thatdoes not interfere with the analytical performance of the sensor butwhich provides the needed fluorescence to distinguish properly alignedlayers as well as identify membrane defects resulting from themanufacturing process.

SUMMARY OF THE INVENTION

The present invention addresses the problems of the prior art machinevision analysis techniques by providing a polymer composition comprisedof fluorescing compounds or residues and articles comprising suchcompositions useful for making a layer of material readily capable ofdetection by using such compositions. Also provided are coumarincompounds useful as fluorophoric markers in the practice of thisinvention. The compositions of the present invention thus provide animproved mechanism for visualizing various constituents of sensors sothat they can be examined and evaluated, and so that the process fortheir manufacture may be optimized, without detracting from theperformance of the sensor.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the severaldrawings, in which like reference numerals are used to indicate likecomponents, in which:

FIG. 1 is a plan view of a plurality of sensor devices embodying thepresent invention;

FIG. 1a is a cross-section of sensor device embodying the presentinvention;

FIGS. 2a and 2b are cross-sectional views of a single sensing element ofthe sensor devices of FIG. 1;

FIG. 3 is a chart representing the results of machine vision inspectionsfrom practicing the present invention;

FIG. 4 is a plan view of a single sensing element of the sensor devicesof FIG. 2b illustrating different registration situations;

FIG. 5 is a cross-sectional view showing alignment dimensions of asingle sensing element of the sensor devices of FIG. 1;

FIGS. 6, 7 and 8 are plan views of a single sensing element of thesensor devices of FIG. 4 illustrating different registration situations;

FIGS. 9a and 9b are schematic views of a machine vision system useful inpracticing this invention; and,

FIG. 10 is a flow chart depicting a method of practicing the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a sensor substrate 20 comprising a number of individualsensor bases 21 arrayed in a regular pattern, the bases 21 being definedby scribing the regular pattern onto a contiguously formed singlemanufacturing piece suitable for handling by automated productionequipment. After manufacturing processes are completed, the sensor bases21 may be separated into a number of individual sensors. Each of thesensor bases 21 has integrated thereon in planar arrangement a singlesensor device 22 comprising a plurality of conductive paths 25 depositedin patterns terminating in electrical contacting pads 23, the conductivepaths 25 originating in sensor elements 30, sensor elements 30 beingcomprised of individual layers 24, 26, and 28 (best seen in FIGS. 2a and2b). The sensor elements 30 are designed to perform either reference ormeasuring tasks and are preferably disposed in banks or rows of linearlydisposed sensor elements with the sensor elements 30 being a referencefunction sensor element 30R being on one side or row and the sensorelements 30 being a detecting or measuring function sensor element 30Mfor the analyte on the other side or row.

In a commercial application, the sensor elements 30 may be usedadvantageously within a chemical analyzer, in combination with a flowchannel member 31 (see FIG. 1a) having grooves 33 positioned over thereference function sensor elements 30R and measuring function sensorelements 30M and joined together at one end of each groove, therebydefining liquid flow channels (not shown). The upper surface of thesensor membrane layer 26 is thusly in fluid and electrolytic contactwith biological sample liquids supplied through the grooves 33. Theamount of analyte in a sample fluid may be determined by using thesensor devices in pairs, with one sensor device being exposed to areference solution containing a known amount of analyte, and the otherbeing exposed to a sample solution containing an unknown amount ofanalyte. U.S. Pat. No. 5,284,568, assigned to the assignee of thepresent invention, is illustrative of such a device. Using well knowncalibration techniques, an assay may be performed in a comparative ordifferential method of measurement to determine the levels of an analytein sample fluids.

As best seen in FIG. 2a, each sensor element 30 comprises a firstdielectric layer 24 and a second dielectric layer 28 formed thereon incontact with a conductive electrode path 25, the layers 24 and 28 havingpatterns of openings therein, the openings in the patterns aligned toform a "well-like" cavity, generally indicated by the letter "D". Asseen in FIG. 2b, a sensor membrane 26 may then be applied within thecavity D formed by openings in the layers 24 and 28 and is positioned incontact with the electrode path 25. The process for making such adevice, preferably using conventional thick film screen printingtechniques and suitable drying means, is well known in the art, forexample, as described in U.S. Pat No. 4,454,007, assigned to theassignee of the present invention. By way of example, one first depositsthe conductor layer 25, typically using a conventional silver conductorpaste, for instance series QS175, available from E. I. du Pont deNemours & Co., Wilmington, Del., then the first dielectric layer 24,typically a conventional ceramic dielectric paste, for instance seriesQS482, the second dielectric layer 28 also for instance series QS482,and finally the sensor membrane layer 26. The purpose of the dielectriclayers 24 and 28 is to establish a cavity D of sufficient depth,generally between 20 and 40 microns, to accommodate the minimum requiredthickness of sensor membrane layer 26 (See FIGS. 2a and 2b). Optionally,an interfacial layer generally composed of a conductive metal andconductive metal-salt compounds may be disposed between the conductorlayer 25 and the sensor membrane layer 26 to stabilize theconductor/membrane interface.

A variety of ion selective membrane compositions may be used for themembrane layer 26, generally comprising an ionophore for an ion ofinterest, a compound capable of dissolving the ionophore and asupporting matrix comprised of one or more binder materials. The matrixcan be any material which is capable of forming a film of sufficientpermeability to produce, in combination with the ionophore and solvent,analyte ion mobility across the film. U.S. Pat. No. 5,401,377 containsinformation about the various chemical constituents and applicableproduction processes useful in production of ISE sensors having anion-sensitive member in direct contact with an electrical conductor andis generally indicative of the state-of-the art.

Useful ionophores include ion carriers such as hemispherands, crownethers, monensin and esters thereof (e.g. methyl-monensin), and othersknown in the art. Ionophores also include ion-exchangers, such aspolymeric ion-exchange materials, and water insoluble salts. The choiceof ionophore will depend on the nature of the ions to be determined,e.g. valinomycin for potassium, methyl-monensin for sodium,tri-n-dodectyl-methylammonium for chloride, etc. The ionophore isdissolved by one or more organic solvents thereby providing sodium ionmobility. If a hydrophobic binder is used as the supporting matrix, thesolvent must be compatible with the binder. The solvent is sometimesidentified in the art as a carrier solvent. Useful solvents includephthalates, sebacates, aromatic and aliphatic ethers, phosphates, mixedaromatic aliphatic phosphonates, adipates, nitrated ethers or esters ormixtures thereof, and others known in the art. The polymeric matrixmaterials were chosen from a variety of substances selected from thegroup consisting of polyvinylchloride, copolymers of polyvinylchloride,polyurethanes, methacrylate polymers, acrylic polymers, and polymerscompatible with polyvinylchloride, and mixtures thereof, withpolyvinylchloride being generally preferred.

Useful membranes including hydrophobic binder materials, an ionophore,and solvating solvents are prepared using known film-coating or castingor screen printing techniques. Materials including synthetic and naturalpolymeric materials, such as poly(vinyl chloride), carboxylatedpoly(vinyl chloride), poly(styrene-co-styrene sulfonic acid), poly(vinylchloride-co-styrene sulfonic acid), poly(vinyl chloride-co-styrenecarboxylic acid) and the like may be used to advantage. High molecularweight poly(vinyl chloride) has been successfully used in the practiceof this invention. Useful plasticizers include 2-ethyl hexyl adipateand/or dioctyl sebacate. One problem encountered with the use of suchpolymeric materials occurs as a consequence of their opticalcharacteristics. In particular, the optical transmissivity of suchpolymeric materials generally falls within a range of values that areessentially transparent to the illuminating systems used in commercialoptical inspection systems. A related problem occurs whenever theoptical reflectivity of the membrane falls within a range of values thatare essentially equivalent to that of the underlying dielectric layersand/or of the substrate material thereby causing a vision system to beunable to reliably differentiate between the membrane and dielectriclayers. The problem of inadequate contrast between layers can beaddressed through the use of optical inspection systems designed todetect fluorescence in combination with membranes modified to fluorescedifferentially from the underlying layers.

The preferred class of fluorophores for this application is the class ofcoumarins. These appear not to interfere with the accuracy of thesensors, compared to fluorophores from the rhodamine or fluoresceinclasses. Within the coumarin class, certain members have been discoveredto have superior fluorescent efficiency that would not be anticipatedfrom their structural similarities or from the spectral or fluorescencedata provided by the vendor's specifications for these products. Thepreferred fluorophores of the present invention are selected from theclass of 7-amino-coumarin derivatives generally having the structureshown below: ##STR1## wherein R₁, R₂, R₃, and R₄ are hydrogen, alkyl oralkylene groups, and R₅, and R₆ are a hydrogen, alkyl, alkylene,haloalkyl, aryl or aromatic, halo, carboxyalkyl, oxo-alkyl, or cyanosubstituent. A preferred fluorophore, coumarin 6(3-(2'-benzothiazolyl)-7-N,N-diethylaminocoumarin), has been discoveredto have superior fluorescence in the membranes without degrading theutility of ISE sensors produced therewith. The structure of coumarin 6is shown below: ##STR2##

Another preferred coumarin is coumarin 314 (1,2,4,5,3H,6H,10H-Tetrahydro-9-carbethoxy(1)benzopyrano(9,9a,1)-gh)quinolizin-10-one)having structure shown below: ##STR3##

Fluorescent Sensor Membranes

Materials

The sensor design, the substrate and the polymeric paste used to preparethe undried membranes were prepared according to the process describedin U.S. Pat. No. 5,522,978 assigned to the assignee of the presentinvention and hereby incorporated by reference. Rhodamine 6G,fluorescein, methylene chloride, isophorone, carboxylated polyvinylchloride, silica, trdodecylmethylammonium chloride, andglycidoxypropyltrimethoxysilane can be obtained commercially from theAldrich Chemical Co. (Milwaukee, Wis.).

The coumarins were obtained commercially from Acros Organics (NewJersey). The sodium ionophore (Fluka III), valinomycin, dioctyl adipate,and potassium tetra(chlorophenyl)borate, can be obtained from FlukaChemika-BioChemika (Ronkonkoma, N.Y.).

The performance of the completed sensor assemblies was tested usingstandard operating protocols on a Dimension® AR clinical chemical systemobtained from Dade Chemistry Systems (Newark, Del.). Ultraviolet spectrawere obtained using an HP model 8452A diode array spectrophotometer,available from Hewlett Packard Co, (Palo Alto, Calif.).

Analytical Performance of ISE Sensors:

Electrolyte testing was done by installing the integrated sensor arrayson a Dimension® system that was equipped with pumps, calibrants, sensorcartridges and appropriate software obtained from Dade Chemistry SystemsInc., Newark, Del. The testing was done by first calibrating thecartridges with two levels of each electrolyte, and then running a panelof test samples consisting of two levels of aqueous bufferedelectrolytes, and three samples of serum based control products. Thecalibrators and verifiers were obtained from Dade Chemistry Systems,Inc., and the control products are Ciba-Corning Co.'s commercialMultiqual® reagents. Medfield, Me. The aqueous buffered samples areDade's commercially available "verifier 1" (V1) and "verifier 2 (V2).The control products had multilevel concentrations of 1, 2, and 3,respectively (MQ1, MQ2, and MQ3). The verifier concentrations weredetermined by comparison to standards based upon gravimetricallydetermined quantities of pure sodium and potassium salts. The controlproduct concentration assignments were determined by comparison tomultiple lots of sensors made without fluorophores.

EXAMPLE 1 Membrane Paste Preparation

1A: Non-fluorescent Paste Compositions:

The pastes used to prepare the ion selective membranes beforeincorporation of an ionophore were made by mixing the ingredients listedin Table 1 below:

                  TABLE 1    ______________________________________              Paste Weight Compositions    Ingredient  Sodium     Potassium                                    Chloride    ______________________________________    Fluka Na III                0.9%       N/A      N/A    Valinomycin N/A        0.9%     N/A    cPVC*       8.4%       8.4%      8.6%    Dioctyl adipate                17.0%      17.0%    N/A    SiO.sub.2   5.1%       5.1%     11.3%    Silane**    3.0%       3.0%      2.9%    Dichloromethane                3.9%       3.9%     N/A    Isophorone  61.6%      61.6%    62.1%    TDMAC***    N/A        N/A      15.0%    Borate****  N/A         0.005%  N/A    ______________________________________     *caboxylated polyvinyl chloride     **glycidoxypropyltrimethoxysilane     ***tridodecylmethylamonium chloride     ****Potassium tetra(chlorophenyl)borate

1B: Fluorescent Paste Composition:

Fluorophores were added to the finished paste compositions of example 1Ato give a concentration of fluorophore equal to 250 μg/g. After removalof the solvents (isophorone and dichloromethane), the concentration was≈710 μg/g in the sodium sensor membrane. The fluorophores shown in Table2 were then evaluated.

                  TABLE 2    ______________________________________    Example     Fluorophore                           Fluorophore Solvent    ______________________________________    1B.1        Rhodamine 6G                           Methanol    1B.2        Fluorescein                           Methanol    1B.3        Coumarin 6 Isophorone    ______________________________________

EXAMPLE 2

2A: Rhodamine Containing Sensors

Sensors were prepared using sodium, potassium and chloride paste thatcontained 250 μg/mL of rhodamine 6G, according to example 1B, Table 2.The results are shown in Table 3 below:

                                      TABLE 3    __________________________________________________________________________    Performance of Sensors with Rhodamine Fluorophore            Slope V1   V2  MQ1  MQ2 MQ3            (mv/decade)                  (mM/L)                       (mM/L)                           (mM/L)                                (mM/L)                                    (mM/L)    __________________________________________________________________________    Na (found)            <50   N/A  N/A N/A  N/A N/A    K (found)             57.58                   2.03                        6.04                            2.75                                 4.31                                     6.73    Cl (found)            -48.17                  94.8  129.08                           81.75                                104.71                                    126.87    Na (assigned)            N/A   120.0                       160.0                           125.31                                154.72                                    184.72    K (assigned)            N/A    2.0  6.0                            2.74                                 4.25                                     6.62    Cl (assigned)            N/A   95.0 128.0                           82.51                                105.06                                    126.66    __________________________________________________________________________

The performance of the sodium sensor was unsatisfactory for therhodamine containing composition, in spite of the low concentration ofthe fluorophore compared to the ionophore. Because of the sensitivity ofthe sensor membrane to its constituents, it was expected that some levelof rhodamine would interfere with the performance of the sodium sensor,it is surprising that the low level tested in this example wouldinterfere as demonstrated. The calibration slope was less than 50mV/decade, compared to a typical response of 57-60 mV/decade when nofluorophore is added. Also, the potassium response was slightly elevatedby at least one standard deviation for the MQ2 and MQ3 results comparedto the values assigned with fluorophore free sensors.

2B: Fluorescein Containing Sensors

Sensors were prepared using sodium, potassium and chloride paste thatcontained 250 μg/mL of fluorescein added as a fluorophore, in a similarmanner as in Example 1B. These sensors were subjected to the same testpanel as in Example 2A using the same verifiers and control products.The results are shown in Table 4 below:

                                      TABLE 4    __________________________________________________________________________    Performance of Sensors with Added Fluorescein Fluorophore            Slope V1   V2  MQ1  MQ2 MQ3            (mv/decade)                  (mM/L)                       (mM/L)                           (mM/L)                                (mM/L)                                    (mM/L)    __________________________________________________________________________    Na (found)            57.79 120.92                       161.12                           130.16                                160.70                                    191.41    K (found)            58.88 2.07 6.02                           2.75 4.27                                    6.65    Cl (found)            -48.61                  95.92                       129.62                           82.07                                104.57                                    126.39    Na (assigned)            N/A   120.0                       160.0                           125.31                                154.72                                    184.72    K (assigned)            N/A   2.0  6.0 2.74 4.25                                    6.62    Cl (assigned)            N/A   95.0 128.0                           82.51                                105.06                                    126.66    __________________________________________________________________________

In all cases, the sensors with added fluorescein gave calibration slopesthat were comparable to what is obtained with sensors having nofluorophore. The sodium sensor gave inaccurate results with the controlproducts MQ1, MQ2, and MQ3. The results were elevated by 5-7 mv/Lcompared to the values assigned with sensors having no fluorophore.

2C: Coumarin 6 Containing Sensors

Sensors were prepared using chloride, potassium, and sodium pastes thatcontained 250 μg/mL of coumarin 6, in the same manner as in example1B.3. These were subjected to a test panel of the same verifiers andcontrol products previously employed. The results are shown in Table 5below:

                                      TABLE 5    __________________________________________________________________________    Performance of Sensors with Added Coumarin 6 Fluorophore            Slope V1   V2  MQ1  MQ2 MQ3            (mv/decade)                  (mM/L)                       (mM/L)                           (mM/L)                                (mM/L)                                    (mM/L)    __________________________________________________________________________    Na (found)            59.7  120.4                       158.8                           123.8                                147.3                                    172.4    K (found)            59.4  2.04 5.94                           2.36 4.12                                    6.31    Cl (found)            -48.7 94.6 128.7                           80.8 102.0                                    126.5    Na (assigned)            N/A   120.0                       160.0                           123  147 172.5    K (assigned)            N/A   2.0  6.0 2.35 4.1 6.28    Cl (assigned)            N/A   95.0 128.0                           80.1 102 126.8    __________________________________________________________________________

All of the sensors gave accurate results with both the verifiers andcontrol products. The calibration slopes were also satisfactory.

EXAMPLE 3

3A: Coumarin Fluorescence:

Samples of fluorophore containing membranes were prepared for spectralstudies. These were made using the formulation for sodium pastedescribed in example 1B, with two changes; the silane coupling agent andthe Fluka III ionophore were omitted. The concentration of fluorophorein the paste was 250 μg/mL.

                  TABLE 6    ______________________________________    Example     Fluorophore                           Fluorophore Solvent    ______________________________________    3A.1        Coumarin 6 Isophorone    3A.2        Coumarin 314                           Isophorone    ______________________________________

3B: Membrane Fluorescence Response:

The paste samples 3A.1 and 3A.2 were dispensed into the preformed wellsof ceramic substrates. Using the apparatus described hereinafter, theundried membranes were illuminated and viewed with a video imagingdevice attached to optical elements. The images were digitized to givepixel responses having 256 gray scale levels. The digitized pixelvalues, indicative of the amount of fluorescence response from theselected fluorophores, are shown as a function of lateral positionacross the membrane in FIG. 3. Starting from the left edge of the graph,the first section of the curves show a low background pixel response ofthe ceramic dielectric where no fluorescent membrane paste was applied.The left hand vertical dotted line indicates the boundary edge of thearea covered with paste. Continuing across the graph, representing thesensor, the pixel scale response rises steeply to a maximum level,reaches a plateau in the area of the maximum membrane thickness, andthen drops to the background response level, at the right side of themembrane.

The response from background lighting in areas not covered with pastegave pixel response values of about 45 units. This was subtracted fromthe values seen in the wet membrane, to give the results shown in FIG.3. The response with example 3A.1, using the preferred coumarin 6surprisingly was about 4× stronger than with the other example, 3A.2.

Sensor Assembly

FIG. 4 is an enlarged and somewhat simplified view of a single sensorelement 30 and, taken with the cross-section shown in FIG. 5, illustratea typical pattern alignment obtained for a single sensor element madeusing the polymeric matrix composition of Table 1 having dispersedtherein an ion exchange ionophore of at least 0.001% and less than about2% by weight of at least one of the preferred coumarin fluorophoresselected from the group of 7-amino-coumarin derivatives as more fullydescribed herein. It is important for proper functioning of the sensorelement 30 that the sensor membrane layer 26 be aligned with and overlapthe dielectric layers 24 and 28 to form an annular ring 32 region ofoverlap that exceeds predetermined minimum dimensions. At the same time,the sensor membrane 26 must be in electrical contact with the underlyingconductive path layer 25. In the instance that the device 21 is sizedapproximately 1 inch by 2 inch, the annular ring 32 region isapproximately an oval having dimensions 0.050 by 0.200 inches, whileoverlap dimensions indicated as "a" and "b" ideally fall into a range,for example from 0.001 to 0.020 inches. The minimum dimensions ofoverlap that are formed by annular ring 32 region are determined asthose overlap dimensions required to provide adequate operatingstability. Inadequate overlap increases the possibility for test fluidscontained in the fluid channel 34 to diffuse underneath sensor membranelayer 26 and to establish erroneous electrical connections directly withthe conductor layer 22.

FIGS. 6, 7 and 8 illustrate several of the various defects that mayoccur from positional inaccuracy of imprinted features. Misregistrationof pattern features that may occur from misalignment of the sensor pastelayers include a pinhole formed in the sensor membrane layer 26indicated by the letter "c" in FIG. 7, incomplete coverage of the sensormembrane layer 26, indicated by the letter "d" in FIG. 6 due tomisregistration and incomplete coverage of the sensor membrane layer 26,indicated by the letter "e" due to incomplete application of the sensormembrane in FIG. 8.

Preferred machine vision systems used in practicing this invention aregenerally comprised of a host computer and special-purpose processinghardware having software implemented applications to make it perform therequired digital image processing operations. Such systems are availablefrom vendors like Omron Electronics (Schaumburg, Ill.), Allen Bradley(Milwaukee, Wis.), and PPT Vision (Minneapolis, Minn.). The principlesinvolved are well known, for example as explained in "Digital ImageProcessing", Gregory A. Baxes, John Wiley & Sons, Inc., New York. Inparticular, image differencing techniques are employed to determinesmall variations between two images that may appear essentially the sameby unaided observation. Using this technique, two images may becompared, pixel by pixel, so that the image portions that are identicalwill subtract to zero (0). Portions of the images that are different,however, will yield a signal other than zero (0). Conventional imageenhancement and analysis techniques may then be applied to determineobject shape measurements that characterize the appearance of an imageaccording to pixel distance around the circumference of the image, pixelarea of the interior of the image, pixel distances of the major andminor axes of image, count of number of holes that exist in the interiorof an image, total pixel area of the holes, and the like. A comparisonof these pixel values with predefined maximum and minimal acceptableabsolute values is used to judge the quality of the membrane sensorlayers. These techniques are well known in the art.

A feature of the present invention is use of dual radiation means toilluminate the sensor to: (1) determine the locations of semi-finishedsensor elements using radiation energy outside the fluorophoreexcitation band; and (2) determine the locations of the correspondingas-deposited sensor membrane paste using radiation energy capable ofexciting the fluorophores included in the sensor membrane 26. Thisallows a comparison between a semi-finished sensor element 30 (like thatshown in FIG. 2a) before the uppermost sensor membrane 26 is applied anda finished sensor element 30 (like that shown in FIG. 2b) after theuppermost sensor membrane 26 is applied. This comparison is accomplishedby utilizing a first illumination means having radiation selected sothat the sensor membrane 30 remains essentially transparent to the imageacquiring means in combination with a second illumination means havingradiation selected so that the sensor membrane 30 becomes essentiallyvisible to the image acquiring means. Appropriate filters to select theradiation without detracting from the images of the sensor membrane 30are included. Means for acquisition, enhancement and analysis of theimages of the sensor element 30 surface image (not shown) comprise aframe-grabber and a microprocessor, and interface means to permitvariable programming of the system's microprocessor-based computer fordesired membrane application and inspection tasks through auser-interactive or computer-controlled system of menus. The system ispreferably combined with a conventional material handling system typicalof piece-parts manufacturing which transports and presents the sensorelements 30 to the machine vision system. These mechanisms, theirinstallation and use are known to those skilled in the art.

FIG. 9a shows such an exemplary sensor production system employingmachine vision for performing the inspection of sensor substrates 21enabled by the sensor membrane compositions of the present invention,the substrates 21 being mounted on a conventional, computer controlled"x-y-z" positioning table 29. A radiation source 13, preferably modelD-7918 obtained from Scholly Fiberoptik GmBH (West Germany), is adaptedto provide a radiation pattern L1 having wavelengths generally in arange from 300 to 700 nm. A first filter 14, for example model 51302produced by Oriel Instruments (Stratford, Conn.) having bandpasscharacteristics such that only radiation having wavelengths greater thanabout 500 nm is passed is positioned proximate to the radiation source13 to intercept and filter radiation pattern L1 so as to illuminate thesemi-finished substrate 21, in particular, the conductor layer 25, andthe two dielectric layers 24 and 28. This filtered radiation pattern L1is distributed from ringlight 12, for instance model 10-1602-03 producedby Ram Optical Inspection (Huntington Beach, Calif.) to illuminate thesensor substrate 21 at an angle from about 20 to 40 degrees relative tothe optical axis defined by a direction perpendicular to the surface ofthe substrate 21.

A shuttle device 9 is adapted to replace the first filter 14 with asecond filter 15, for example model 57530 produced by Oriel, the secondfilter 15 having narrow bandpass filter characteristics such that onlyradiation having wavelengths preferably centered between about 400 and500 nm to provide illumination selected to excite the coumarinfluorophores contained in the sensor membrane 26. The filtered radiationL1 is emitted from ringlight 12 to illuminate the sensor substrate 21 atan angle from about 20 to 40 degrees relative to the optical axis.

A radiation emission filter 17, for example model 51302 produced byOriel having bandpass filter characteristics selected such that onlyradiation having those wavelengths greater than the representativeexcitation wavelengths of coumarin fluorophores, preferably betweenabout 400 and 800 nm, is positioned before the image acquiring means 18to intercept the radiation emitted by an excited fluorophore. Thus, onlythat radiation having wavelengths emitted by the tagging fluorophorewithin the sensor membrane 26 are incident upon image acquiring means 18when the second filter 15 is in use. Image acquiring means 18 preferablycomprises a high resolution, solid state, MOS (metal oxidesemiconductor) type with asynchronous frame reset capability, forinstance model XC77 produced by Sony Corporation (Toko, Japan) equippedwith appropriate optical elements. This capability allows the imageacquiring means 18 to capture the image of a sensor element 30 withspatial resolution of approximately 0.0003 inches per pixel. Achangeover for other sizes/shapes of sensor elements 30 may beaccommodated by simply adjusting the vertical positions of the imageacquiring means 18 and different optical element 11.

In an alternate embodiment, shown in FIG. 9b, a second illuminatingsource 16, for example model 50-3500-00 produced by Ram OpticalInspection is employed to provide a beam of radiation generally in awavelength range from 550 to 650 nm through a beamsplitter 15, forinstance model 0102020 produced by Esco Products (Oak Ridge, N.J.)positioned in the optical axis with the split portion of the beamcaptured by image acquiring means 18 and the reflected portion beingnormally incident upon the substrate. If used in combination with thepreferred arrangement shown in FIG. 9a, this alternate arrangementallows the optimum combination of angularly incident and normallyincident radiation upon the substrate from either of the two sources 13and 16 to provide the highest degree of image contrast and capture,depending upon the surface roughness and optical adsorption/reflectioncharacteristics of the sensor substrate 21 and layers 24, 25, 26, and28. The arrangement and management of the electronic circuitry of theimage acquiring means 18 and the frame-grabber 19 are widely known andthe routines of comparing the various images are also well-known.

FIG. 10 is a flowchart of a process for determining the target area onsemi-finished sensor substrates 21 for sensor membrane layer 26application, for detecting application flaws within the sensor membranelayer 26 and misregistration of the applied sensor membrane layer 26.The process is further adapted to provide correction information tocomputer 22. In this process, information regarding the desiredpositioning of the sensor membrane is determined by illuminating thesemi-finished substrate 21 like shown in FIG. 2a in which only theconductor layer 25 and first and second dielectric layers 24 and 28 havebeen printed onto the sensor substrate 21 with radiation that has beenfiltered through bandpass filter 14, indicated by step 102. The imageacquiring means 18 thus acquires an image of the top surface of thesemi-finished sensor element so that the location may be determined ofeach of the cavities D (FIG. 2a) defined by the dielectric layers 24 and28 where a sensor membrane 26 is to be applied (FIG. 2b), as shown inboxes 102 and 104.

After the computer 22 determines the precise location of the cavity D, amembrane paste application operation is initiated and the membraneapplication means (not shown) applies a predetermined amount and patternof sensor membrane paste within the cavity D as depicted in box 106.Conventional alignment techniques are employed to control theapplication means to apply the membrane paste in proper alignment on thesensor element 21 so that each cavity D is in communication with no morethan one of the conductive elements 25, the membrane 26 being portionedand disposed within said openings so that communication is establishedbetween said membrane portion 26 and the conductive elements 25.Application of the membrane composition takes place using dispensingtechniques, with equipment available from vendors such as Asymtek(Carlsbad, Calif.), Otto Engineering (Carpentersville, Ill.) and CamalotSystems (Haverhill, Me.).

In the subsequent membrane inspection mode, a sensor element 21 to beinspected is illuminated using radiation in the excitation region of thefluorophore so that the image capture system acquires an image of thesensor membrane 26 as applied to the sensor element 30 and depicted inbox 108. The computer 22 and frame-grabber 19 utilized in this inventionprovides capability for analysis of the digital images in box 110.Consequently, the computer 22 is able to analyze the image of the sensormembrane 26 with respect to the previously generated image in box 102 ofthe cavity D as depicted in box 112 to determine the degree ofcoincident alignment by making a conventional flaw and dimensionanalysis between the two images arising from errors in the applicationof the sensor membrane 26 as depicted in box 114. Subsequentconventional alignment techniques are employed to control theapplication of the membrane paste in proper alignment on the sensorelement 30 as depicted in box 116.

Applicants have thus discovered that, notwithstanding the performancesensitivity of the ISE sensor membrane to its chemical constituents,certain fluorophores have been found that may be advantageously added tothe composition of the sensor membrane 26 without interfering with theperformance of chloride, potassium, or sodium sensors. There are twoprimary requirements for the fluorophore which must be met in such afluorophore design: first, it must not interfere with the analyticalperformance of the sensor by causing greater than one percent deviationof the performance of the incorporating membrane; and, second, it mustbe sufficiently fluorescent within the membrane to providedistinguishable optical contrast relative to the background portion ofthe sensor under ambient lighting.

The first requirement of the fluorophore is that it cause less than onepercent deviation of the amount of measured analyte relative to theanalyte concentration determined with compositions having no fluorophoretherein. There is general understanding of the compositionalrequirements of a usable sensor membrane; however, the effects ofchanges in composition and specific concentrations or chemicalstructures for the components is not well understood. Since ISE sensorsare known to be highly sensitive to the composition of the surface layerof the membrane, it is not obvious or understood why some fluorophoreswill interfere and others will not, especially at the relatively lowconcentration of fluorophore used in the Examples herein. Thefluorophore is at a concentration of 0.025% in the paste, whereas thespecific ionophore for sodium and potassium is ≈1% so that the ionophoreis approximately 40× higher in concentration than is the fluorophore.The presence of an ionic species in the membrane of ISE sensors isexpected to have an adverse effect on the performance of the sensor.However, it is surprising that at a concentration of less than 2.5% ofthe active ion-carrier, there is a detectable degradation ofperformance. Even at this low concentration, the rhodamine andfluorescein examples hereinabove show unexpected, and un-acceptableinterference in the sodium sensor. These results are contrary to thoseexpected from the prior art. In the prior art, some ionic species areexpected to improve performance. For example, in the case offluorescein, a lipophilic anion, the expected effect is an improvementin specificity, based upon theory described by Lindner, Erno, et al. in"Response of Site-controlled, Plasticized Membrane Electrodes.",Analytical Chemistry, 60 (1988): 295-301. Even at this lowconcentration, the rhodamine and fluorescein examples hereinbelow showunexpected, and un-acceptable interference in the sodium sensor. Whilethe amino coumarins carry no charge in the native state, side reactionscould occur between them and other components of the sensor membranethat would yield ionic products that would interfere with sensorperformance. It is not obvious that the amino coumarins are sufficientlychemically inert to be free of such side reactions.

Secondly, a sufficiently high concentration of fluorophore is requiredwithin the membrane since the membrane must capture a sufficient amountof the excitation light source to emit an amount of light to permitaccurate discrimination between its fluorescence and the backgroundimage due to reflected illumination from stray ambient light sources.Unfortunately, the fluorescence of fluorophores is known to be sensitiveto self-quenching at the concentration levels required to meet theseconditions. Self-quenching is a general term within the art thatdescribes the phenomenon of decreasing fluorescence quantum yield seenas fluorophore concentrations are increased. The processes responsiblefor this self-quenching are understood in principle, but again, not wellenough to accurately anticipate the usefulness of specific compositions.

Another difficulty faced in selection of the fluorophore is that inaddition to self-quenching, non-specific chemical processes take placeduring bonding between the fluorophore and the silane coupling agentused to provide covalent linkage of the membrane to the dielectric ofthe sensor coupling agent. These non-specific chemical processesgenerally act to reduce the yield of fluorescence an unpredictableamount.

Surprisingly, certain fluorophores have been successfully incorporatedinto the sensor membrane 26 as shown in the preceding Examples.Compounds from the coumarin class of fluorophores have proven effectivein rendering a detectable fluorescent emission from the normallytransparent sensor pastes as described hereinafter without causinggreater than one percent interference with the electrochemical responseof the membrane. The dyes used in the Examples are all stronglyfluorescent when measured at low concentrations in common solvents. Forexample, the quantum yields for fluorescence for the two coumarinsdescribed above are 85% for coumarin 6, and 77% for coumarin 314,(Eastman Laser Products Dataservice Publication JJ-169, 1977, KodakOptical Products, Eastman Kodak Company, Rochester, N.Y.) Rhodamine andfluorescein compositions were expected to be useful in a ISE sensor,based on the fluorescence obtainable therewith at a low concentration;however, experimental tests described above unexpectedly determined thatthey caused greater than one percent errors in the analyticalperformance of the sensors at these low concentrations.

It is within the scope of the present invention to employ other sensormembrane compositions to enhance visualization of various constituentsof ion selective electrodes. For instance, by using two differentfluorophores in two membrane layers applied successively in a lower andupper relationship, an optical comparison of the relative alignment andintegrity of the two layers can be made using essentially the same imageacquisition and analysis techniques described herein. Alternatively, byusing a fluorophore in the lower layer, and using a light absorbingagent capable of substantially blocking the incident radiation in theupper layer, the relative alignment and integrity of the two layers maybe determined using the image acquisition and analysis techniquesdescribed herein. The agent, for example an inert phthalocyaninepigment, must also cause less than one percent deviation upon theperformance of the membrane layer.

It is to be understood that the embodiments of the invention disclosedherein are illustrative of the principles of the invention and thatother modifications may be employed which are still within the scope ofthe invention. Accordingly, the present invention is not limited tothose embodiments precisely shown and described in the specification.

What is claimed is:
 1. A method of assembling an ion selective devicefor sensing the presence of an analyte, the device comprising a planarsubstrate having in sequence thereon, a plurality of conductiveelements, at least one dielectric layer having a plurality of openingsforming a cavity therethrough, and a membrane comprising a polymericmatrix, an ion exchange ionophore, and less than 0.001% and 2% by weightof at least one fluorophore, said fluorophore being essentiallyunaffected by the presence of said analyte,said fluorophore beingselected from the class of 7-amino-coumarin derivatives having structureshown below: ##STR4## where R₁, R₂, R₃, and R₄ are hydrogen, alkyl oralkylene groups, and R₅, and R₆ are hydrogen, alkyl, alkylene,haloalkyl, aryl, halo, carboxyalkyl, oxo-alkyl, or cyano, the membraneportioned and disposed within said openings, the method comprising:applying the plurality of conductive elements onto the substrateapplying at least one dielectric layer over the conductive elements;applying the membrane within the openings formed within the dielectriclayer so that communication is established between said membraneportions and said conductive elements; and, using a machine visionmeasurement apparatus adapted for determining the positioning of theopenings formed within the dielectric layer and controlling the applyingof the membrane within the openings, the machine vision measurementapparatus having illumination means to firstly determine the locationsof the openings using radiation having energy outside the excitationband of said fluorophore and to secondly determine the locations of themembrane within the openings using radiation having energy capable ofexciting the fluorophore included in the sensor membrane, so that theprocess for applying of the membrane within the openings may beoptimized and controlled.